The present invention relates to the field of virology. Embodiments described herein include cDNA clones of the Langat tick-borne flavivirus.
There are more than 60 antigenically related, positive strand RNA viruses in the arthropod-borne flavivirus genus of the family Flaviviridae, many of which are important human pathogens. The antigenically-related tick-borne encephalitis virus complex of the flavivirus family includes tick-borne encephalitis virus (TBEV, formerly called Russian spring-summer encephalitis virus), Kyassanur forest disease, Langat, Louping ill, Negishi, Omsk hemorrhagic fever, and Powassan viruses (Calisher, C. H., Karabatsos, N., Datrymple, J. M., Shope, R. E., Porterfield, J., Westaway, E. G., and Brant, W. E. (1989) Antigenic relationships between flaviviruses are determined by cross-neutralization test with polyclonal antisera. J. Gen. Virol., 70, 27-43.; Monath , T. P., and Heinz, F. X. (1996) Flaviviruses. In xe2x80x9cFields Virology.xe2x80x9d (B. N. Fields, D. M. Knipe and P. M. Howley, Eds.), 3rd ed., pp. 961-1035. Lippincott-Raven Publishers, Philadelphia and New York.). These viruses are endemic throughout most of the Northern Hemisphere, and except for Langat, cause human disease of varying severity that can have mortality as high as 20 to 30%. Tick-borne encephalitis remains a pressing public health problem in Eastern Europe and Russia, where 9,000-12,000 patients are diagnosed annually. A significant increase in mortality was recorded in 1956 and 1964, when morbidity reached 4,500-4,600 per 100,000 persons (Gaidamovich, S. Y. (1995) Tick-borne flavivirus infections. In xe2x80x9cExotic Viral Infections.xe2x80x9d (J. S. Porterfield, Ed.) pp. 203-221. Chapman and Hall, London.).
The tick-borne encephalitis flaviviruses share envelope glycoprotein epitopes that often induce cross-resistance among viruses of the group. Approximately three decades ago, these properties of antigenic cross reactivity and the subsequent recognition of virulence polymorphism suggested that successful immunization might be achieved using a live, naturally attenuated tick-borne flavivirus (Il""enko, V. I., Smorodincev, A. A., Prozorova, I. N., and Platonov, V. G. (1968) Experience in the study of a live vaccine made from the TP21 strain of Malayan Langat virus. Bull. W. H. O. 39, 425-431.; Price, W. H., Thind, I. S., Teasdall, R. D., and O""Leary, W. (1970) Vaccination of human volunteers against Russian spring-summer (RSS) virus complex with attenuated Langat E5 virus. Bull. W. H. O. 42, 89-94.; Mayer, V., Orolin, D., Pogady, J., Starek, M., Kubistova, K., Gajdo-Sova, E., and Buran, I. (1976) Experimental live tick-borne encephalitis vaccine (Langat E5xe2x80x314xe2x80x3 virus clone): volunteers 1 and 2 years after single-dose immunization. Acta virol., 20, 215-225.). The impetus for this approach was the recovery of a virus from ticks in Malaysia, namely Langat virus (LGT), strain TP21, that did not appear to be associated with human disease under natural conditions (Gordon Smith, C. E. (1956) A virus resembling Russian spring-summer encephalitis virus from an Ixodid in Malaya. Nature (London) 178, 581-582.). Immunization of animals and human volunteers with LGT induced a high level of virus-neutralizing antibodies against various members of TBEV complex such as Powassan, Kyassanur forest disease and TBEV (Price, W. H., Thind, I. S., Teasdall, R. D., and O""Leary, W. (1970) Vaccination of human volunteers against Russian spring-summer (RSS) virus complex with attenuated Langat E5 virus. Bull. W. H. O. 42, 89-94.; Price, W. H., and Thind, I. S. (1973) Immunization of mice against Russian spring-summer virus complex and monkeys against Powassan virus with attenuated Langat E5 virus. Am. J. Trop. Med. Hyg. 22, 100-108.). Nonetheless, TP21 exhibited neurovirulence and neuroinvasiveness (xe2x80x9cperipheral virulencexe2x80x9d) when tested in mice and therefore was considered too dangerous for use as a vaccine candidate. (Gordon Smith, C. E. (1956) A virus resembling Russian spring-summer encephalitis virus from an Ixodid in Malaya. Nature (London) 178, 581-582.; Thind, I. S., and Price, W. H. (1966a) A chick embryo attenuated strain (TP21 E5) of Langat virus. I. Virulence of the virus for mice and monkeys. Am. J. Epidemiol., 84, 193-213.; Pletnev, A. G., and Men, R. (1998) Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4. Proc. Natl. Acad. Sci. USA, 95, 1746-1751.). Notwithstanding the direct neurovirulence measured by intracerebral inoculation observed for the TP21 Langat virus, its peripheral virulence (neuroinvasiveness) was significantly less than that of the very virulent Far Eastern strains of TBEV that produces human disease that has a 20 to 30% mortality. Several LGT strains, which were partially attenuated for mice and monkeys, were isolated and studied in the USA, Russia and Czechoslovakia (Nathanson, N., Thind, I. S., O""Leary, W., and Price, W. H. (1968) Histological studies of the monkey neurovirulence of group B arboviruses. IV. Evaluation of an attenuated strain (E5) of Langat virus. Am. J. Epidemiol. 88, 103-112.; Price, W. H., Thind, I. S., Teasdall, R. D., and O""Leary, W. (1970) Vaccination of human volunteers against Russian spring-summer (RSS) virus complex with attenuated Langat E5 virus. Bull. W. H. O. 42, 89-94.; Mayer, V., Orolin, D., Pogady, J., Starek, M., Kubistova, K., Gajdo-Sova, E., and Buran, I. (1976) Experimental live tick-borne encephalitis vaccine (Langat E5xe2x80x314xe2x80x3 virus clone): volunteers 1 and 2 years after single-dose immunization. Acta virol., 20, 215-225.; Smorodincev, A. A., and Dubov, A. V. (1986) Live vaccines against tick-borne encephalitis. In xe2x80x9cTick-Borne Encephalitis and Its Vaccine Prophylaxisxe2x80x9d, (A. A. Smorodincev, ed.), pp. 190-211. Meditsina, Leningrad.). One such strain, designated Yelantsev, was studied extensively in over 600,000 vaccinees in Russia as an experimental live vaccine against TBEV during the early 1970""s (Smorodincev, A. A., and Dubov, A. V. (1986) Live vaccines against tick-borne encephalitis. In xe2x80x9cTick-Borne Encephalitis and Its Vaccine Prophylaxisxe2x80x9d, (A. A. Smorodincev, ed.), pp. 190-211. Meditsina, Leningrad.). Studies were discontinued when it was learned that vaccination was associated with a very low frequency of encephalitis, approximately one case per 20,000 immunizations. Nonetheless, this experience confirmed the initial view that LGT was highly attenuated and dearly the most benign member of the tick-borne flavivirus complex.
Shortly thereafter, a more attenuated mutant of LGT, designated strain E5, was selected by 42 passages in embryonated chicken eggs. LGT E5 exhibited less virulence for mice and monkeys than its TP21 parent. More recently, a study demonstrated that E5 exhibited less neurovirulence in mice than its TP21 parent (Pletnev, A. G., and Men, R. (1998) Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4. Proc. Natl. Acad. Sci. USA, 95, 1746-1751.). Also, unlike its TP21 parent, E5 exhibited very little neuroinvasiveness and this was detectable only in a small fraction of mice inoculated peripherally with the largest amount of virus possible. Before considering the more attenuated E5 mutant of LGT as a possible candidate for use in prophylaxis of severe human disease caused by certain members of tick-borne flavivirus group, in the interest of safety scientists must reduce or ablate the last vestiges of virulence of LGT TP21 and E5 for mice by using a strategy that has been employed successfully in the past to attenuate dengue virus, namely the introduction of site-specific mutations into the full length infectious cDNA of the virus. Thus, there is a need for full length infectious cDNA clones of Langat.